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. 2016 Mar 31:7:91.
doi: 10.3389/fphar.2016.00091. eCollection 2016.

The Anti-fibrotic Actions of Relaxin Are Mediated Through a NO-sGC-cGMP-Dependent Pathway in Renal Myofibroblasts In Vitro and Enhanced by the NO Donor, Diethylamine NONOate

Chao Wang  1 , Barbara K Kemp-Harper  1 , Martina Kocan  2 , Sheng Yu Ang  2 , Tim D Hewitson  3 , Chrishan S Samuel  1
Affiliations

The Anti-fibrotic Actions of Relaxin Are Mediated Through a NO-sGC-cGMP-Dependent Pathway in Renal Myofibroblasts In Vitro and Enhanced by the NO Donor, Diethylamine NONOate

Chao Wang et al. Front Pharmacol. .

Abstract

Introduction: The anti-fibrotic hormone, relaxin, has been inferred to disrupt transforming growth factor (TGF)-β1/Smad2 phosphorylation (pSmad2) signal transduction and promote collagen-degrading gelatinase activity via a nitric oxide (NO)-dependent pathway. Here, we determined the extent to which NO, soluble guanylate cyclase (sGC) and cyclic guanosine monophosphate (cGMP) were directly involved in the anti-fibrotic actions of relaxin using a selective NO scavenger and sGC inhibitor, and comparing and combining relaxin's effects with that of an NO donor.

Methods and results: Primary renal cortical myofibroblasts isolated from injured rat kidneys were treated with human recombinant relaxin (RLX; 16.8 nM), the NO donor, diethylamine NONOate (DEA/NO; 0.5-5 μM) or the combined effects of RLX (16.8 nM) and DEA/NO (5 μM) over 72 h. The effects of RLX (16.8 nM) and DEA/NO (5 μM) were also evaluated in the presence of the NO scavenger, hydroxocobalamin (HXC; 100 μM) or sGC inhibitor, ODQ (5 μM) over 72 h. Furthermore, the effects of RLX (30 nM), DEA/NO (5 μM) and RLX (30 nM) + DEA/NO (5 μM) on cGMP levels were directly measured, in the presence or absence of ODQ (5 μM). Changes in matrix metalloproteinase (MMP)-2, MMP-9 (cell media), pSmad2 and α-smooth muscle actin (α-SMA; a measure myofibroblast differentiation) (cell layer) were assessed by gelatin zymography and Western blotting, respectively. At the highest concentration tested, both RLX and DEA/NO promoted MMP-2 and MMP-9 levels by 25-33%, while inhibiting pSmad2 and α-SMA expression by up to 50% (all p < 0.05 vs. untreated and vehicle-treated cells). However, 5μM of DEA/NO was required to produce the effects seen with 16.8 nM of RLX over 72 h. The anti-fibrotic effects of RLX or DEA/NO alone were completely abrogated by HXC and ODQ (both p < 0.01 vs. RLX alone or DEA/NO alone), but were significantly enhanced when added in combination (all p < 0.05 vs. RLX alone). Additionally, the direct cGMP-promoting effects of RLX, DEA/NO and RLX+DEA/NO (which all increased cGMP levels by 12-16-fold over basal levels; all p < 0.01 vs. vehicle-treated cells) were significantly inhibited by pre-treatment of ODQ (all p < 0.05 vs. the respective treatments alone).

Conclusion: These findings confirmed that RLX mediates its TGF-β1-inhibitory and gelatinase-promoting effects via a NO-sGC-cGMP-dependent pathway, which was additively augmented by co-administration of DEA/NO.

Keywords: cGMP; cell signaling; fibrosis; myofibroblasts; nitric oxide; relaxin; transforming growth factor-β1.

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Figures

FIGURE 1
FIGURE 1
The effects of RLX vs. DEA/NO on pSmad2, α-SMA, MMP-2 and MMP-9. Shown are (A) representative (duplicate samples from one experiment) Western blots of pSmad2 and α-SMA; and (B) gelatin zymographs of latent (L) and active (A) MMP-2 and MMP-9 from untreated control rat renal myofibroblasts and cells treated with vehicle (10 μM NaOH) alone, RLX (16.8 nM) alone or increasing concentrations of DEA/NO (0.1–5 μM) alone, after 72 h in culture. Also shown are the mean ± SEM optical density (OD) of (A) pSmad2 and α-SMA (corrected for Smad2/3 loading); and (B) (latent and active) MMP-2 and MMP-9, as determined from densitometry measurements of the Western blots or zymographs. Numbers in parenthesis represent the number of independent experiments carried out in duplicate. *P < 0.05, **P < 0.01 vs. respective groups highlighted.
FIGURE 2
FIGURE 2
The combined effects of RLX and DEA/NO on pSmad2, α-SMA, MMP-2 and MMP-9. Shown are (A) representative (duplicate samples from one experiment) Western blots of pSmad2 and α-SMA; and (B) gelatin zymographs of latent (L) and active (A) MMP-2 and MMP-9 from vehicle (10 μM NaOH)-treated rat renal myofibroblasts and cells treated with RLX (16.8 nM) alone, DEA/NO (5 μM) alone or the combined effects of RLX (16.8 nM) alone and DEA/NO (5 μM) after 72 h in culture. Also shown are the mean ± SEM OD of (A) pSmad2 and α-SMA (corrected for Smad2/3 loading); and (B) (latent and active) MMP-2 and MMP-9, as determined from densitometry measurements of the Western blots or zymographs. Numbers in parenthesis represent the number of independent experiments carried out in duplicate. **P < 0.01, ***P < 0.001 vs. respective groups highlighted.
FIGURE 3
FIGURE 3
The effects of HXC and ODQ on basal and DEA/NO-mediated changes of pSmad2, α-SMA, MMP-2 and MMP-9. Shown are (A) representative (duplicate samples from one experiment) Western blots of pSmad2 and α-SMA; and (B) gelatin zymographs of latent (L) MMP-2 and MMP-9 from vehicle (10 μM NaOH)-treated rat renal myofibroblasts and cells treated with HXC (100 μM), ODQ (5 μM) alone, DEA/NO (5 μM) alone or the combined effects of DEA/NO (5 μM) and HXC (100 μM) or ODQ (5 μM), after 72 h in culture. Also shown are the mean ± SEM optical density (OD) of (A) pSmad2 and α-SMA (corrected for Smad2/3 loading); and (B) (latent) MMP-2 and MMP-9, as determined from densitometry measurements of the Western blots or zymographs. Numbers in parenthesis represent the number of independent experiments carried out in duplicate. **P < 0.01 vs. respective groups highlighted.
FIGURE 4
FIGURE 4
The effects of HXC and ODQ on RLX-mediated changes of pSmad2, α-SMA, MMP-2 and MMP-9. Shown are (A) representative (duplicate samples from one experiment) Western blots of pSmad2 and α-SMA; and (B) gelatin zymographs of latent (L) MMP-2 and MMP-9 from untreated (control) rat renal myofibroblasts and cells treated with RLX (16.8 nM) alone or the combined effects of RLX (16.8 nM) and HXC (100 μM) or ODQ (5 μM), after 72 h in culture. Also shown are the mean ± SEM optical density (OD) of (A) pSmad2 and α-SMA (corrected for Smad2/3 loading); and (B) (latent) MMP-2 and MMP-9, as determined from densitometry measurements of the Western blots or zymographs. Numbers in parenthesis represent the number of independent experiments carried out in duplicate. **P < 0.01 vs. respective groups highlighted.
FIGURE 5
FIGURE 5
The effects of ODQ on RLX ± DEA/NO-mediated changes in cGMP accumulation. Shown are the mean ± SEM cGMP levels in rat renal myofibroblasts treated for 30 min with RLX (30 nM), DEA/NO (5 μM), or the combined effects of both; and sub-groups of correspondingly treated cells that were pre-exposed to ODQ (5 μM) for 15 min prior to administration of RLX + DEA/NO. Numbers in parenthesis represent the number of independent experiments carried out. *P < 0.05, **P < 0.01 vs. respective groups highlighted.
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